Bottom Line:
L-arabinose was confirmed to be a negative effector of BtAraR.In the structure of the BtAraR-DNA complex, we found the unique interaction of arginine intercalating its guanidinum moiety into the base pair stacking of B-DNA.L-arabinose binding induces movement of wHTH domains, resulting in a conformation unsuitable for DNA binding.

Figure 1: Comparative genomics reconstruction of AraR regulons in Bacteroides spp. (A) Maximum likelihood phylogenetic tree of NrtR family regulators from the Bacteroidetes phylum. The AraR and XylR regulators from Bacteroides thetaiotaomicron (33% identity to each other, shown in green boxes) belong to two different branches of the NrtR family regulators in the Bacteroidetes. Two previously characterized ADP-ribose-responsive NrtR regulators from Shewanella oneidensis and Synechocystis spp. are shown in pink boxes. Proteins with solved crystal structures are shown in red frames. (B) Sequence logo for AraR-binding motif in Bacteroides spp. The logo was constructed using ∼50 candidate AraR sites from 17 Bacteroides spp. (C) Multiple sequence alignment of promoter regions of BT0356 (araM) and its orthologs in Bacteroides spp. Tandem AraR-binding sites and a putative binding site of a Crp-like regulator are highlighted. (D) AraR-regulated metabolic pathway for utilization of L-arabinose and its polymers. The pathway includes extracytoplasmic hydrolytic enzymes (Abn, Abf), transporters through the outer membrane (SusCD) and the inner membrane (AraP), periplasmic L-arabinose mutarotase (AraM) and cytoplasmic L-arabinose catabolic enzymes (AraA, AraB, AraD). (E) Reconstructed AraR regulons in three Bacteroides genomes. Candidate AraR-binding sites are shown by red circles; sequences of AraR sites in these and other genomes are listed in Supplementary Table S1. Genes/potential operons are shown by arrows grouped in large white boxes. Genes with the same functional roles are marked in matching colors. Hypothetical genes are shown by gray and white arrows. Hybrid two-component system (HTCS) regulators and their candidate binding sites are shown by black arrows and squares, respectively. Transcriptional induction of AraR-controlled operons by L-arabinose and arabinose-containing polymers is summarized using the previous transcriptomics studies (41,42).

Mentions:
Orthologs of the B. thetaiotaomicron AraR (BT0354) and XylR (BT0791) regulators were identified only within the Bacteroidetes phylum. AraR is present in 17 Bacteroides and 15 Prevotella spp., whereas XylR was found in 25 Bacteroides spp. and in several other Bacteroidetes (Figure 1A). We noted a strong tendency of araR and xylR genes to cluster on the chromosome with the arabinose and xylose utilization genes, respectively. Among 30 non-redundant Bacteroides species analyzed in this work, the AraR regulators and arabinose catabolic pathway genes were found in 17 species, while the other 13 Bacteroides species potentially have lost the arabinose catabolic genes. Multiple alignments of these AraR proteins revealed high overall conservation of their primary sequences (Supplementary Figure S1), suggesting the AraR orthologs are functionally identical with potentially preserved specificities toward the effector molecule and DNA sites. The Nudix signature motif GX5-EX7REUXEEXGU (where U is a hydrophobic residue and X is any residue) is strictly conserved in all known active Nudix hydrolases, but it is impaired in the NrtR regulators of NAD metabolism, several of which are known to be enzymatically inactive (15). Similarly to NrtR regulators, the Nudix signature motif is not conserved in the AraR proteins from Bacteroidetes, where two or three glutamate residues are substituted with other amino acids (Supplementary Figure S1). These observations suggest that AraR regulators are also enzymatically inactive and utilize their Nudix hydrolase-like domains for ligand binding.

Figure 1: Comparative genomics reconstruction of AraR regulons in Bacteroides spp. (A) Maximum likelihood phylogenetic tree of NrtR family regulators from the Bacteroidetes phylum. The AraR and XylR regulators from Bacteroides thetaiotaomicron (33% identity to each other, shown in green boxes) belong to two different branches of the NrtR family regulators in the Bacteroidetes. Two previously characterized ADP-ribose-responsive NrtR regulators from Shewanella oneidensis and Synechocystis spp. are shown in pink boxes. Proteins with solved crystal structures are shown in red frames. (B) Sequence logo for AraR-binding motif in Bacteroides spp. The logo was constructed using ∼50 candidate AraR sites from 17 Bacteroides spp. (C) Multiple sequence alignment of promoter regions of BT0356 (araM) and its orthologs in Bacteroides spp. Tandem AraR-binding sites and a putative binding site of a Crp-like regulator are highlighted. (D) AraR-regulated metabolic pathway for utilization of L-arabinose and its polymers. The pathway includes extracytoplasmic hydrolytic enzymes (Abn, Abf), transporters through the outer membrane (SusCD) and the inner membrane (AraP), periplasmic L-arabinose mutarotase (AraM) and cytoplasmic L-arabinose catabolic enzymes (AraA, AraB, AraD). (E) Reconstructed AraR regulons in three Bacteroides genomes. Candidate AraR-binding sites are shown by red circles; sequences of AraR sites in these and other genomes are listed in Supplementary Table S1. Genes/potential operons are shown by arrows grouped in large white boxes. Genes with the same functional roles are marked in matching colors. Hypothetical genes are shown by gray and white arrows. Hybrid two-component system (HTCS) regulators and their candidate binding sites are shown by black arrows and squares, respectively. Transcriptional induction of AraR-controlled operons by L-arabinose and arabinose-containing polymers is summarized using the previous transcriptomics studies (41,42).

Mentions:
Orthologs of the B. thetaiotaomicron AraR (BT0354) and XylR (BT0791) regulators were identified only within the Bacteroidetes phylum. AraR is present in 17 Bacteroides and 15 Prevotella spp., whereas XylR was found in 25 Bacteroides spp. and in several other Bacteroidetes (Figure 1A). We noted a strong tendency of araR and xylR genes to cluster on the chromosome with the arabinose and xylose utilization genes, respectively. Among 30 non-redundant Bacteroides species analyzed in this work, the AraR regulators and arabinose catabolic pathway genes were found in 17 species, while the other 13 Bacteroides species potentially have lost the arabinose catabolic genes. Multiple alignments of these AraR proteins revealed high overall conservation of their primary sequences (Supplementary Figure S1), suggesting the AraR orthologs are functionally identical with potentially preserved specificities toward the effector molecule and DNA sites. The Nudix signature motif GX5-EX7REUXEEXGU (where U is a hydrophobic residue and X is any residue) is strictly conserved in all known active Nudix hydrolases, but it is impaired in the NrtR regulators of NAD metabolism, several of which are known to be enzymatically inactive (15). Similarly to NrtR regulators, the Nudix signature motif is not conserved in the AraR proteins from Bacteroidetes, where two or three glutamate residues are substituted with other amino acids (Supplementary Figure S1). These observations suggest that AraR regulators are also enzymatically inactive and utilize their Nudix hydrolase-like domains for ligand binding.

Bottom Line:
L-arabinose was confirmed to be a negative effector of BtAraR.In the structure of the BtAraR-DNA complex, we found the unique interaction of arginine intercalating its guanidinum moiety into the base pair stacking of B-DNA.L-arabinose binding induces movement of wHTH domains, resulting in a conformation unsuitable for DNA binding.